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Arquivos Brasileiros de Endocrinologia & Metabologia

On-line version ISSN 1677-9487

Arq Bras Endocrinol Metab vol.54 no.8 São Paulo Nov. 2010 



Monoallelic thyroid peroxidase gene mutation in a patient with congenital hypothyroidism with total iodide organification defect


Mutação monoalélica no gene da tireoperoxidase em paciente com hipotireoidismo congênito com defeito total de incorporação de iodeto



Solange Caires NevesI; Paola Rossi MezaliraI; Vera M. A. DiasII; Antonio J. ChagasII; Maria VianaII; Hector TargovnikIII; Meyer KnobelI; Geraldo Medeiros-NetoI; Ileana G. S. RubioIV

IThyroid Unit, Cellular and Molecular Endocrinology Laboratory, LIM-25, Faculdade de Medicina da Universidade de São Paulo (FMUSP), SP, Brazil
IICenter for Actions and Research in Diagnostic Support (NUPAD), Faculdade de Medicina, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil
IIILaboratorio de Biología Molecular, Cátedra de Genética y Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina
IVBiological Sciences Departament, Universidade Federal de São Paulo (Unifesp), São Paulo, SP, Brazil

Correspondence to




The aim of this study was to identify the genetic defect of a patient with dyshormonogenetic congenital hypothyroidisms (CH) with total iodide organification defect (TIOD). A male child diagnosed with CH during neonatal screening. Laboratory tests confirmed the permanent and severe CH with TIOD (99% perchlorate release). The coding sequence of TPO, DUOX2, and DUOXA2 genes and 2957 base pairs (bp) of the TPO promoter were sequenced. Molecular analysis of patient's DNA identified the heterozygous duplication GGCC (c.1186_1187insGGCC) in exon 8 of the TPO gene. No additional mutation was detected either in the TPO gene, TPO promoter, DUOX2 or DUOXA2 genes. We have described a patient with a clear TIOD causing severe goitrous CH due to a monoallelic TPO mutation. A plausible explanation for the association between an autosomal recessive disorder with a single TPO-mutated allele is the presence of monoallelic TPO expression.


O objetivo deste estudo foi identificar defeitos genéticos em paciente com hipotireoidismo congênito (HC) por disormonogênese e defeito total de incorporação de iodeto (DIIT). Neonato do sexo masculino com HC diagnosticado pelo rastreamento neonatal. Exames clínicos e radiológicos confirmaram que o paciente apresentava HC severo e permanente com DIIT (teste de perclorato: 99%). A região codificadora dos genes TPO, DUOX2, DUOXA2 e 2957 pares de bases (pb) do promotor de TPO foram sequenciados. No paciente foi identificada a duplicação em heterozigose GGCC no éxon 8 do gene TPO (c.1186_1187insGGCC). Nenhuma outra mutação foi localizada nos genes TPO, incluindo o promotor, DUOX2 ou DUOXA2. Descrevemos paciente com grave defeito de organificação de iodeto, provocando HC severo com bócio, em consequência de uma única mutação monoalélica no gene TPO. A expressão monoalélica no tecido tireoideano explicaria a associação de uma doen­ça autossômica recessiva com uma única mutação monoalélica.




Congenital hypothyroidism (CH) is the most common cause of preventable mental retardation with prevalence ranging from 1:2000 to 1:3000 newborns. In recent years prevalence has increased due to lower TSH cutoff values (10 mU/mL) used in the newborn screening programs (1).

Confirmatory biochemical exams associated with imaging and perchlorate tests are performed when the child reaches three years of age. The tests are conducted after four weeks of L-thyroxine withdrawal, when there is no risk of brain damage.

The most common cause of congenital hypothyroidism is thyroid dysgenesis (athyreosis, hemiagenesis, hypoplasia, and ectopia) seen in approximately 75% of the cases. Defects in thyroid hormone synthesis, or dyshormogenesis, accounts for 15%-20% of the cases and is caused by defects in genes involved in the thyroid-hormone synthesis pathway (2). Mutations in thyroid peroxidase (TPO) are the most frequent genetic alterations in dyshormonogenesis, with prevalence of 1/40,000 newborns (3). Mutations in thyroglobulin (TG) (4), sodium iodide symporter (NIS), pendrin (PDS), dual oxidase 2 (DUOX2) (5), dual oxidase maturation factor 2 (DUOXA2) (6), and iodotyrosine deiodinase (DEHAL1) (7) genes have been also identified in patients with dyshormonogenesis.

TPO is a heme-containing enzyme localized on the apical membrane of the follicular cell. TPO catalyzes the iodination of tyrosine residues in thyroglobulin, and couples these residues to form thyroid hormones (8).

TPO mutations have been associated with total iodide organification defect (TIOD), where more than 80% of iodide is discharged from the intrathyroidal iodide pool, indicating that the iodide that is taken up was not incorporated into the TG protein. Partial iodide organification defects are associated with iodide discharge levels of between 25% and 79% (9). More than 60 TPO gene mutations have been previously described. Linkage studies have characterized the autosomal recessive mode of inheritance of TPO gene disorders (3).

The aim of this study was to identify the genetic defect of a patient with dyshormonogenetic CH and TIOD.



The index patient is a boy who was diagnosed with CH during neonatal screening with serum TSH of 35.9 µUI/mL. His parents were not consanguineous. He was started on a daily dose of L-thyroxine (12.5 mg/kg) at 30 days of life. CH was confirmed at the age of 3 years, when the medication was suspended for 4 weeks and laboratory tests showed: serum TSH: 635.3 µUI/mL (reference value: 0.5 – 4.5 µUI/mL); total T4 (T4T): 1.0 µg/dL (reference value: 4 – 11 µg/dL), Free T4 (FT4): 0.1 ng/dL (reference value: 0.7 – 1.7 ng/dL); TG: 129.6 ng/mL (reference value: 5 − 30 ng/mL) and iodide discharge after perchlorate: 99% (reference value < 15%). The scintigraphy and ultrasound examinations indicated a heterogeneous eutopic, larger than normal thyroid gland.

This study was approved by the Ethical Committee of the Hospital das Clínicas, University of São Paulo Medical School. Informed consent was obtained from the parents of the patient.



Thyroid function tests

Serum Total T4 (TT4), free T4 (FT4), total T3, TSH, and TG levels were determined by electrochemiluminescence immunoassay (Roche Corporation, Indianapolis, IN). Perchlorate discharge test was performed with 25 mCi of radiodine 131I administered orally and thyroid uptake was measured after 1 hour. At this point 0.5 g of potassium perchlorate was orally administered and sequential uptakes were measured at 15-minute intervals for an additional hour. The effect of perchlorate was expressed as a percentage of radioiodine uptake at 1 hour. Thyroid volume was calculated after echographic studies. Scintigraphy was performed with 131I.

DNA sequencing

Peripheral blood DNA from the patient, his parents and his brother was isolated by the sodium dodecyl sulfate/proteinase K method. The complete coding sequence of the human TPO gene, including splice sites and flanking intronic regions of each exon, was amplified using primers and conditions reported previously (9). A TPO promoter region of 2957 bp was also sequenced with specific primers (Table 1).



DNA sequencing of each amplified fragment was performed with a ABI 377 system (Applied Biosystems Corp., Foster City, CA) using the same TPO-specific primers used in the amplification step. The sequences were compared with those of the human TPO gene sequence (GeneID: 7173).

In order to amplify and sequence the coding region and intron-exon borders of the DUOX2 and DUOXA2 gene we used the primers and conditions previously described (5,6). These sequences were compared with those of human DUOX2 and DUOXA2 (GeneID: 50506 and GeneID: 405753, respectively).

A control group of 100 healthy subjects without thyroid disease was evaluated to determine whether the observed DNA substitutions were bona fide mutations or polymorphisms. All control subjects had normal FT4 (0.7-1.5 ng/dL) and TSH levels (0.5-4.5 U/mL).



Screening for TPO gene mutations identified the known insertion of 4 base pairs GGCC in position 1186 of the cDNA (c.1186_1187insGGCC) (or in position 1227 of the DNA gene), in exon 8, which introduces a new stop codon in exon 9 (p.Ala396fsX472) (10). This mutation was present in a heterozygous form (Figure 1) and was the only mutation identified in the TPO gene. Polymorphisms were also found in the promoter, intronic and coding sequences of the TPO gene (Table 2).





The 33 exons of the DUOX2 gene were sequenced revealing only polymorphisms in this gene. These DUOX2 alterations were also found in more than 1% of the control subject's DNAs (Table 3). The sequence analysis of DUOXA2 gene did not show sequence alterations in the patient's DNA.



The c.1186_1187insGGCC mutation was not identified in his mother, stepfather or brother's DNA. Polymorphism's analysis confirmed that there was not genetic relation between the patient and the stepfather.



TPO enzymatic activity is essential for thyroid hormonogenesis. Inactivating TPO mutations are the most frequent molecular basis for dyshormonogenetic congenital goitrous hypothyroidism due to iodide organification defect (3).

The most frequently studied phenotype is the TIOD, characterized by iodide discharge after perchlorate > 80%. Most affected patients present with severe and permanent hypothyroidism with mental retardation if not treated in the neonatal period. Discharge levels between 25% and 79% are denominated partial iodide organification defects (PIOD). The variety of clinical features in PIOD patients suggests that diverse mechanisms such as delayed or reduced activity of enzymes involved in hormonogenesis, or defects in iodine storage and release (11) may lead to PIOD.

The established molecular basis of TIOD is either due to biallelic (homozygous or compound heterozygous) inactivation mutations in the TPO gene, or biallelic inactivation mutations in the DUOX2 gene. Biallelic inactivation of DUOX2 results in a complete block of H2O2 generation, a relatively rare genotype (5). More frequent, are the inactivating mutations in the TPO gene resulting in a TIOD (3).

Few studies have described cases of PIOD due to TPO biallelic mutations (9,12,13). Mutations in DUOX2 and DUOXA2 have also been reported in PIOD patients (5,6). Inactivating monoallelic DUOX2 mutations have also been reported in relation to transient forms of relatively mild hypothyroidism (5).

In this study we described a male child with severe congenital goitrous hypothyroidism and TIOD, extremely low serum FT4 and TT4 coexisting with very high levels of serum TSH and TG. Moreover a high iodine uptake and 99% of iodide discharge after perchlorate confirmed the absence of organification of iodide. Molecular analysis of the patient's DNA indicated presence of duplication of the tetranucleotide GGCC (c.1186_1187insGGCC) in exon 8 of the TPO gene, in heterozygous state. Interestingly, this was the only mutation identified in the whole TPO coding region, in the intron-exon borders and in ~3000bp of the TPO promoter. No additional mutation was found either in DUOX2 or DUOXA2 genes.

This mutation has been previously identified in other Brazilian TIOD patients as well as in other reports (3). The resulting frameshift introduces a new stop codon in exon 9, which could result in a grossly truncated protein and promote a drastic reduction of TPO activity in the affected thyroid tissue (Figure 2) (10).

Cases with a typical TIOD phenotype and a single TPO-mutated allele have also been described. The frequency of this phenomenon is unexpectedly high. It has been found in around 9% to 65% of the reported TIOD or PIOD patients of different geographic origin (9,14-18). In our experience, three out of four TIOD Brazilian patients and three out of six PIOD patients, all with TPO mutations, had only one mutant allele (data not shown).

Genetic explanations for monoallelic TPO mutations in TIOD patients may be the presence of intronic mutations creating an alternative splicing site or of mutations located in the distal promoter, more than 3000 bp upstream of the transcription initiation site. The presence of large gene deletions has also been advocated. Polymorphisms, however, both in the coding and noncoding regions of TPO patient's gene deny this possibility.

Other mechanisms that may explain our's and other's TIOD cases with a single TPO-mutated allele is the presence of monoallelic expression in the thyroid tissue, as has been shown by Fugazzola and cols. (18). This group reported one family with TIOD due to monoallelic expression of a maternal mutant TPO allele in the thyroid tissue.

In diploid eukaryotic organisms, there are three different classes of monoallelically expressed genes. One class is the autosomal imprinted genes whose monoallelic expression is regulated in a parent-of-origin-specific manner. A second class is X-inactivated genes regulated by a random process. The third class is autosomal genes subject to random monoallelic expression (such as odorant receptor, immunoglobulins, T-cell receptors, interleukins, and natural killer cell receptors) (19). A recent study has shown that this last class of genes is relatively frequent. Using a general strategy for genome-wide analysis of monoallelic expression, Gimelbrant and cols. (19) showed that ~ 5% to 10% of 4000 assessed autosomal genes display random monoallelic transcription in human cells with stability in each clonal cell line.

Unusual regulatory mechanisms have been observed for monoallelically expressed genes: e.g., DNA rearrangement and allele-specific DNA methylation for the immunoglobulin gene; a single enhancer associated with multiple odorant receptor gene promoters located on different chromosomes (19). Other transcriptional or post-transcriptional mechanisms may also be considered, such as alternative polyadenylation, chromatin remodeling, histone modifications, RNA edditing and microRNA regulation.

The mechanism resulting in monoallelic TPO expression may also be independent of parental origin. Fugazzola and cols. (18) detected the maternal mutant TPO allele expression associated to the lack of paternal allele transcripts at the thyroid tissue level. In our study, despite the fact that we were not able to study the biological father's DNA, the exclusive expression of the paternal mutated allele may be considered. The stochastic origin of monoallelic TPO expression may be also supported by the detection of the same mutant TPO allele in the healthy mother and in her severe congenital hypothyroid daughter with TIOD (data not shown). This was the case of another patient studied in our laboratory with monoallelic TPO mutation associated with silencing of the paternal allele.

In conclusion we have described a patient with 99% of iodide discharged after perchlorate with a clear organification defect causing severe congenital goitrous hypothyroidism due to a monoallelic TPO mutation (c.1186_1187insGGCC) in exon 8.

Acknowledgments: We acknowledge the financial support of FAPESP (grant 99/03570-3) and Instituto da Tiroide, São Paulo, SP, Brazil.

Disclosure: no potential conflict of interest relevant to this article was reported.



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Correspondence to:
Ileana G. S. Rubio
Rua Prof. Artur Riedel, 275
09972-270 − Diadema, SP, Brazil

Received on Jul/30/2010
Accepted on Nov/23/2010

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